Shikonin attenuates cerebral ischemia/reperfusion injury via inhibiting NOD2/RIP2/NF-κB-mediated microglia polarization and neuroinflammation.

OBJECTIVES: Microglia-mediated neuroinflammation plays a crucial role in the pathophysiological process of multiple neurological disorders such as ischemic stroke, which still lacks effective therapeutic agents. Shikonin possesses anti-inflammatory and neuroprotective properties. However, its underlying mechanism remains elusive. This study aimed to investigate whether Shikonin confers protection against cerebral ischemia/reperfusion (I/R) injury by modulating microglial polarization and elucidate the associated mechanisms. METHODS: This study employed an oxygen-glucose deprivation and reoxygenation (OGD/R) BV2 microglial cellular model and a middle cerebral artery occlusion/reperfusion (MCAO/R) animal model to investigate the protection and underlying mechanism of Shikonin against ischemic stroke. RESULTS: The results demonstrated that Shikonin treatment significantly reduced brain infarction volume and improved neurological function in MCAO/R rats. Simultaneously, Shikonin treatment significantly reduced microglial proinflammatory phenotype and levels of proinflammatory markers (inducible-NO synthase (iNOS), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), and IL-6), increased microglial anti-inflammatory phenotype and levels of anti-inflammatory markers (Arginase-1 (Arg1), transforming growth factor-beta (TGF-ß), and IL-10), reversed the expression of Nucleotide-binding oligomerization domain 2 (NOD2) and phosphorylation receptor interacting protein 2 (p-RIP2), and suppressed nuclear factor kappa-B (NF-κB) signaling activation in the ischemic penumbra regions. These effects of Shikonin were further corroborated in OGD/R-treated BV2 cells. Furthermore, overexpression of NOD2 markedly attenuated the neuroprotective effects of Shikonin treatment in MCAO/R rats. NOD2 overexpression also attenuated the regulatory effects of Shikonin on neuroinflammation, microglial polarization, and NF-κB signaling activation. CONCLUSION: This study illustrates that Shikonin mitigates inflammation mediated by microglial proinflammatory polarization by inhibiting the NOD2/RIP2/NF-κB signaling pathway, thereby exerting a protective role. The findings uncover a potential molecular mechanism for Shikonin in treating ischemic stroke.

Receptor Interacting Ser/Thr-Protein Kinase 2 as a New Therapeutic Target.

Receptor interacting serine/threonine protein kinase 2 (RIPK2) is a downstream signaling molecule essential for the activation of several innate immune receptors, including the NOD-like receptors (NOD1 and NOD2). Recognition of pathogen-associated molecular pattern proteins by NOD1/2 leads to their interaction with RIPK2, which induces release of pro-inflammatory cytokines through the activation of NF-κB and MAPK pathways, among others. Thus, RIPK2 has emerged as a key mediator of intracellular signal transduction and represents a new potential therapeutic target for the treatment of various conditions, including inflammatory diseases and cancer. In this Perspective, first, an overview of the mechanisms that underlie RIPK2 function will be presented along with its role in several diseases. Then, the existing inhibitors that target RIPK2 and different therapeutic strategies will be reviewed, followed by a discussion on current challenges and outlook.

NOD1 cooperates with HAX-1 to promote cell migration in a RIPK2- and NF-ĸB-independent manner.

The human Nod-like receptor protein NOD1 is a well-described pattern-recognition receptor (PRR) with diverse functions. NOD1 associates with F-actin and its protein levels are upregulated in metastatic cancer cells. A hallmark of cancer cells is their ability to migrate, which involves actin remodelling. Using chemotaxis and wound healing assays, we show that NOD1 expression correlated with the migration rate and chemotactic index in the cervical carcinoma cell line HeLa. The effect of NOD1 in cell migration was independent of the downstream kinase RIPK2 and NF-ĸB activity. Additionally, NOD1 negatively regulated the phosphorylation status of cofilin, which inhibits actin turnover. Co-immunoprecipitation assays identified HCLS1-associated protein X-1 (HAX-1) as a previously unknown interaction partner of NOD1. Silencing of HAX-1 expression reduced the migration behaviour to similar levels as NOD1 knockdown, and simultaneous knockdown of NOD1 and HAX-1 showed no additive effect, suggesting that both proteins act in the same pathway. In conclusion, our data revealed an important role of the PRR NOD1 in regulating cell migration as well as chemotaxis in human cervical cancer cells and identified HAX-1 as a protein that interacts with NOD1 and is involved in this signalling pathway.

RIPK2 promotes the progression of colon cancer by regulating BIRC3-mediated ubiquitination of IKBKG.

Colon cancer is a cancer with high morbidity and mortality worldwide. Receptor interacting serine/threonine kinase 2 (RIPK2) has been identified as a proto-oncogene, but its role in colon cancer is largely unknown. Herein, we found that RIPK2 interference could inhibit the proliferation and invasion of colon cancer cells, and promote apoptosis. Baculoviral IAP repeat containing 3 (BIRC3) is an E3 ubiquitin ligase, which was found highly expressed in colon cancer cells. Co-immunoprecipitation (Co-IP) experiments showed that RIPK2 could directly bind with BIRC3. Then, we demonstrated that RIPK2 overexpression promoted the expression of BIRC3, BIRC3 interference could eliminate RIPK2-dependent cell proliferation and invasion, and BIRC3 overexpression rescued the suppressive effect of RIPK2 interference on cell proliferation and invasion. We further identified IKBKG, an inhibitor of nuclear factor kappa B, as a ubiquitination substrate targeted by BIRC3. IKBKG interference could eliminate the inhibitory effect of BIRC3 interference on cell invasion. RIPK2 could promote BIRC3-mediated ubiquitination of IKBKG, inhibit the expression of IKBKG protein, and promote the expression of NF-κB subunits p50 and p65 proteins. In addition, DLD-1 cells transfected with sh-RIPK2 or/and sh-BIRC3 were injected into mice to establish a tumor xenograft model, and we found that administration of sh-RIPK2 or sh-BIRC3 impeded the growth of xenograft tumors in vivo, and co-administration displayed a better inhibitory effect. In general, RIPK2 promotes the progression of colon cancer by promoting BIRC3-mediated ubiquitination of IKBKG and activating the NF-κB signaling pathway.

Card9 protects sepsis by regulating Ripk2-mediated activation of NLRP3 inflammasome in macrophages.

Sepsis is characterized by systemic inflammation, it's caused by primary infection of pathogenic microorganisms or secondary infection of damaged tissue. In this study, we focus on sepsis-induced intestine barrier functional disturbalice, presenting as increased permeability of intestinal epithelium. We observed that the phenotype of LPS-induced sepsis was exacerbated in Card9-/- mice, especially displaying more serious intestinal inflammation and gut barrier dysfunction. Next, we found the hyperactivation of NLRP3 inflammasome in the intestinal macrophages of Card9-/--sepsis mice. Moreover, Card9 over-expression decreased NLRP3 inflammasome activation in macrophages. Furthermore, we found that Card9 inhibited NLRP3 inflammasome activation by recruiting Ripk2. The competitive binding between Ripk2 with Caspase-1, instead of ASC with Caspase-1, inhibited the NLRP3 inflammasome activation. Over-expression of Ripk2 alleviated septic intestinal injury caused by Card9 deficiency. Taken together, we suggested Card9 acts as a negative regulation factor of NLRP3 inflammasome activation, which protects against intestinal damage during sepsis. Therefore, maintaining Card9-Ripk2 signaling homeostasis may provide a novel therapy of septic intestinal damage.

Pan-cancer analysis reveals RIPK2 predicts prognosis and promotes immune therapy resistance via triggering cytotoxic T lymphocytes dysfunction.

BACKGROUND: Receptor-interacting protein kinase 2 (RIPK2, also known as RIP2) was reported to be associated with bacterial infections as well as inflammatory responses. However, the role of RIPK2 in prognosis and immunotherapy response is yet to be elucidated in human pan-cancer. METHODS: In this study, we investigated the expression, gene alteration landscape and prognostic value of RIPK2 in 33 cancers through various databases including Ualcan, cBioportal and Gene Expression Profiling Interactive Analysis 2 (GEPIA2). Then, the correlation between RIPK2 and immune infiltration, immune score, stromal score, and ESTIMATE score was investigated in the Cancer Genome Atlas (TCGA) and tumor immune estimation resource (TIMER) databases. Independent cohorts were utilized to explore the role of RIPK2 in tumor immunotherapy response. Furthermore, Gene set enrichment analysis (GSEA) was conducted to explore the mechanisms by which RIPK2 regulates immune therapy resistance. Single-cell RNA-seq datasets were used to analyze the expression level of RIPK2 on different immune cells. Moreover, CellMiner database was used to explore the relationship between RIPK2 expression with drug response. RESULT: Compared with normal tissue, tumor tissue had a higher expression level of RIPK2 in various cancers. Survival analysis showed that high expression of RIPK2 associated with poor prognosis in numerous cancers. RIPK2 was found to promote a series of immune cell infiltration and B cells, macrophages, and neutrophils were significantly positively correlated with the expression of RIPK2. Moreover, RIPK2 affected immune score, stromal score and ESTIMATE score for a wide range of cancers. In the vast majority of 33 cancers, gene co-expression analysis showed that RIPK2 was positively correlated with the expression of immune checkpoint markers, such as PDCD1 (PD-1), CD274 (PD-L1), CTLA4 and TIGIT. RIPK2 aggravated cytotoxic T lymphocyte (CTL) dysfunction and related to the poor efficacy of immune checkpoint blockade in skin cutaneous melanoma (SKCM) and kidney renal clear cell carcinoma (KIRC). High expression of RIPK2 promoted innate immunotherapy resistance and adaptive immunotherapy resistance through IL-6/JAK/STAT3 signaling, interferon-gamma response, and interferon-alpha response pathway. CONCLUSIONS: These results confirmed that RIPK2 could serve as a prognostic biomarker and promoted immune therapy resistance via triggering cytotoxic T lymphocytes dysfunction.

High expression of RIPK2 is associated with Taxol resistance in serous ovarian cancer.

BACKGROUND: Taxol resistance in serous ovarian cancer is responsible for its poor prognosis, yet the underlying mechanism is still poorly understood. Thus, we probed the mechanism of Taxol resistance in serous ovarian cancer with multiple bioinformatic methods to provide novel insights into potential therapies. METHODS: The differentially expressed genes (DEGs) in Taxol-sensitive and Taxol-resistant cell lines and their relationship with the overall survival (OS) and progression-free interval (PFI) of ovarian cancer patients were analyzed using gene expression datasets from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). The role of receptor interacting serine/threonine kinase 2 (RIPK2) was validated via identification of its coexpressed genes, functional analysis and generation of a protein-protein interaction (PPI) network. The single sample gene set enrichment analysis (ssGSEA) was used to explore immune infiltration, and genomic alterations of RIPK2 were also analyzed via cBio Cancer Genomics Portal (cBioProtal). RESULTS: RIPK2 was highly expressed in Taxol resistant ovarian cancer cell lines, and its high expression was also linked with shorter OS and PFI in serous ovarian cancer patients. The PPI network analysis and pathway analysis demonstrated that RIPK2 might participate in the positive regulation of NF-κB transcription factor activity. RIPK2 expression was related to tumor microenvironment alterations, which might participate in the formation of Taxol resistance. CONCLUSIONS: Our studies suggested that high expression of RIPK2 is related to Taxol resistance in serous ovarian cancer, and that RIPK2 induces Taxol resistance through NOD1/RIPK2/NF-κB inflammatory pathway activation and tumor microenvironment changes.

RIP2-deficiency induces inflammation in response to SV40 Large T induced genotoxic stress through altered ROS homeostasis.

Deciphering signaling pathways that regulate the complex interplay between inflammation and cell death is a key challenge in understanding innate immune responses. Over recent years, receptor interacting protein (RIP) kinases have been described to regulate the interplay between inflammation and cell death. Whereas RIP1 and 3, the most well described members of the RIP kinase family, play important roles in necroptosis, RIP2's involvement in regulating inflammation, cell death processes and cancer is less well described and controversially discussed. Here, we demonstrate that RIP2 exerts immune regulatory functions by regulating mitochondrial damage and mitochondrial superoxide production in response to SV40 LT-induced genotoxic stress by the induction of ULK1-phosphorylation, therefore regulating the expression of interferon stimulated genes (ISGs) and NLRP3-inflammasome dependent IL-1ß release. Because RIP2 is upregulated and/or activated in autoimmune/inflammatory disease and cancer, observations from this study promise implications of RIP kinases in human disease.

A pancancer analysis of the carcinogenic role of receptor-interacting serine/threonine protein kinase-2 (RIPK2) in human tumours.

BACKGROUND: To explore the expression and carcinogenic mechanism of RIPK2 in human tumours, and to provide the theoretical basis for the further study of RIPK2. METHODS: We used the TCGA, CPTAC, HPA databases to analyse the expression, mutation, and prognosis of RIPK2 in human tumours. Through the Cbioportal, Ualcan, TIMER2.0, and STRING websites, We understand the genetic variation, immune infiltration and enrichment analysis of RIPK2 related genes. RESULTS: RIPK2 was highly expressed in most tumours (such as BRCA, COAD and LUSC, etc.), and the high expression of RIPK2 was correlated with tumour stage and prognosis. In addition, Amplification was the main type of RIPK2 in tumour mutation state, and the amplification rate was about 8.5%. In addition, RIPK2 was positively associated with tumour-infiltrating immune cells (such as CD8+ T, Tregs, and cancer-associated fibroblasts). According to the KEGG analysis, RIPK2 may play a role in tumour mainly through NOD-like signaling pathway and NF-kappaB signaling pathway. GO enrichment analysis showed that the RIPK2 is mainly related to I-kappaB kinase/NF-kappaB signaling, Ribonucleoprotein granule and Ubiquitin-like protein ligase binding. CONCLUSION: RIPK2 plays an important role in the occurrence, development and prognosis of malignant tumours. Our pancancer study provided a relatively comprehensive description of the carcinogenic effects of RIPK2 in different tumours, and provided useful information for further study of RIPK2.

Receptor-interacting protein kinase 2 (RIPK2) stabilizes c-Myc and is a therapeutic target in prostate cancer metastasis.

Despite progress in prostate cancer (PC) therapeutics, distant metastasis remains a major cause of morbidity and mortality from PC. Thus, there is growing recognition that preventing or delaying PC metastasis holds great potential for substantially improving patient outcomes. Here we show receptor-interacting protein kinase 2 (RIPK2) is a clinically actionable target for inhibiting PC metastasis. RIPK2 is amplified/gained in ~65% of lethal metastatic castration-resistant PC. Its overexpression is associated with disease progression and poor prognosis, and its genetic knockout substantially reduces PC metastasis. Multi-level proteomics analyses reveal that RIPK2 strongly regulates the stability and activity of c-Myc (a driver of metastasis), largely via binding to and activating mitogen-activated protein kinase kinase 7 (MKK7), which we identify as a direct c-Myc-S62 kinase. RIPK2 inhibition by preclinical and clinical drugs inactivates the noncanonical RIPK2/MKK7/c-Myc pathway and effectively impairs PC metastatic outgrowth. These results support targeting RIPK2 signaling to extend metastasis-free and overall survival.

Fusobacterium nucleatum promotes esophageal squamous cell carcinoma progression via the NOD1/RIPK2/NF-κB pathway.

Fusobacterium nucleatum, found in the oral cavity, influences the progression of gastrointestinal cancers. Additionally, our previous results suggested that F. nucleatum is associated with poor patient prognosis in esophageal squamous cell carcinoma (ESCC). However, the mechanism by which F. nucleatum affects aggressive tumor behavior has yet to be elucidated. We have conducted this clinical, in vitro, and in vivo study to clarify the mechanism of ESCC progression induced by F. nucleatum. Transmission electron microscopy revealed that F. nucleatum invaded and occupied ESCC cells and impacted gene and protein expression. Comprehensive mRNA expression and pathway enrichment analyses of F. nucleatum-treated ESCC cells identified the "NF-κB" and "NOD-like receptor" signaling pathways as enriched. We confirmed the relationship between the presence of F. nucleatum and NF-κB activation in resected ESCC tissues. Furthermore, F. nucleatum-treated ESCC cells demonstrated enhanced growth ability, and NF-κB activation, as well as overexpression of NOD1 and phosphorylated RIPK2. Furthermore, treated cells showed accelerated tumor growth, with NF-κB activation in xenograft models. F. nucleatum invaded ESCC cells and induced the NF-κB pathway through the NOD1/RIPK2 pathway, leading to tumor progression.

Activation of RIPK2-mediated NOD1 signaling promotes proliferation and invasion of ovarian cancer cells via NF-κB pathway.

The goal of this study was to investigate the role and mechanism of action of nucleotide oligomerization domain receptor 1 (NOD1) in ovarian cancer. Results showed that the expressions of NOD1 and receptor interacting serine/threonine kinase 2 (RIPK2) were notably upregulated in non-metastatic and metastatic ovarian tumors compared with matched non-tumor tissues, and their expression in metastatic tumor tissues was higher than that in non-metastatic tumors. Overexpression of NOD1 facilitated the expression of proliferation-related proteins (PCNA and Ki67) and proliferation and invasion of ovarian cancer cells. Overexpression of NOD1 promoted NF-κB expression and phosphorylation. Importantly, NOD1 bound with RIPK2, and silencing of RIPK2 partly rescued the promotion of NOD1 to NF-κB expression and its phosphorylation. The promotion of NOD1 to ovarian cancer cell proliferation and invasion was partly reversed by RIPK2 silencing. Results from our in vivo study indicate that overexpression of NOD1 accelerated the growth of ovarian cancer tumors, expression of proliferation-related proteins, and activation of NF-κB. However, silencing of NOD1 suppressed tumor growth. In summary, NOD1 facilitates ovarian cancer progression by activating NF-κB signaling by binding to RIPK2. We suggest a new strategy for the treatment of ovarian cancer.

The potent and selective RIPK2 inhibitor BI 706039 improves intestinal inflammation in the TRUC mouse model of inflammatory bowel disease.

Mouse and human data implicate the NOD1 and NOD2 sensors of the intestinal microbiome and the associated signal transduction via the receptor interacting protein kinase 2 (RIPK2) as a potential key signaling node for the development of inflammatory bowel disease (IBD) and an attractive target for pharmacological intervention. The TRUC mouse model of IBD was strongly indicated for evaluating RIPK2 antagonism for its effect on intestinal inflammation based on previous knockout studies with NOD1, NOD2, and RIPK2. We identified and profiled the BI 706039 molecule as a potent and specific functional inhibitor of both human and mouse RIPK2 and with favorable pharmacokinetic properties. We dosed BI 706039 in the spontaneous TRUC mouse model from age 28 to 56 days. Oral, daily administration of BI 706039 caused dose-responsive and significant improvement in colonic histopathological inflammation, colon weight, and terminal levels of protein-normalized fecal lipocalin (all P values <0.001). These observations correlated with dose responsively increasing systemic levels of the BI 706039 compound, splenic molecular target engagement of RIPK2, and modulation of inflammatory genes in the colon. This demonstrates that a relatively low oral dose of a potent and selective RIPK2 inhibitor can modulate signaling in the intestinal immune system and significantly improve disease associated intestinal inflammation.NEW & NOTEWORTHY The RIPK2 kinase at the apex of microbiome immunosensing is an attractive target for pharmacological intervention. A low oral dose of a RIPK2 inhibitor leads to significantly improved intestinal inflammation in the murine TRUC model of colitis. A selective and potent inhibitor of the RIPK2 kinase may represent a new class of therapeutics that target microbiome-driven signaling for the treatment of IBD.

Excretory/secretory proteins of adult Toxocara canis induce changes in the expression of proteins involved in the NOD1-RIP2-NF-κB pathway and modulate cytokine production in mouse macrophages.

Dog roundworm (Toxocara canis) is the major causative agent of toxocarosis, a parasitic disease of both veterinary and medical importance. Knowledge gaps in fundamental and applied aspects hinder the control of this important zoonotic disease. To have a better understanding of Toxocara infection and host immune responses, mouse macrophages were exposed to excretory/secretory (ES) proteins released by adult worms of T. canis in vitro. The messenger RNA transcription and protein expression of nucleotide-binding oligomerization domain-containing protein 1 (NOD1), receptor interacting protein 2 (RIP2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in macrophages were analysed using quantitative real-time PCR (qRT-PCR) and Western blot. The levels of tumour necrosis factor alpha (TNF-ɑ), interleukin-1 beta (IL-1ß) and IL-6 released by the stimulated macrophages were analysed using enzyme-linked immunosorbent assay. It was found that 20 µg/mL ES proteins of adult T. canis induced the expression of NOD1, RIP2 and NF-κB in mouse macrophages at both transcriptional and translational levels after 9 h of incubation in vitro. Incubation with 20 µg/mL ES proteins also modulated the production of pro-inflammatory cytokines TNF-ɑ, IL-1ß and IL-6 by the macrophages. Taken together, ES proteins of adult T. canis appeared to be able to affect the macrophage NOD1-RIP2-NF-κB signalling pathway, which might play a role in regulating the production of proinflammatory cytokines. Further investigation of these aspects should lead to a better understanding of immune recognition of and modulation by Toxocara canis in host animals.

The E3 ubiquitin ligase RNF186 and RNF186 risk variants regulate innate receptor-induced outcomes.

Balancing microbial-induced cytokines and microbial clearance is critical at mucosal sites such as the intestine. How the inflammatory bowel disease (IBD)-associated gene RNF186 regulates this balance is unclear. We found that macrophages from IBD-risk rs6426833 carriers in the RNF186 region showed reduced cytokines to stimulation through multiple pattern recognition receptors (PRRs). Upon stimulation of PRRs, the E3-ubiquitin ligase RNF186 promoted ubiquitination of signaling complex molecules shared across PRRs and those unique to select PRRs. Furthermore, RNF186 was required for PRR-initiated signaling complex assembly and downstream signaling. RNF186, along with its intact E3-ubiquitin ligase activity, was required for optimal PRR-induced antimicrobial reactive oxygen species, reactive nitrogen species, and autophagy pathways and intracellular bacterial clearance in human macrophages and for bacterial clearance in intestinal myeloid cells. Cells transfected with the rare RNF186-A64T IBD-risk variant and macrophages from common rs6426833 RNF186 IBD-risk carriers demonstrated a reduction in these RNF186-dependent outcomes. These studies identify mechanisms through which RNF186 regulates innate immunity and show that RNF186 IBD-risk variants demonstrate a loss of function in PRR-initiated outcomes.

RIPK2 is an unfavorable prognosis marker and a potential therapeutic target in human kidney renal clear cell carcinoma.

Receptor Interacting Serine/Threonine Kinase 2 (RIPK2) is located on chromosome 8q21 and encodes a protein containing a C-terminal caspase activation and recruitment domain (CARD), which is a component of signaling complexes in both the innate and adaptive immune pathways. To estimate the value of RIPK2 in evaluating the prognosis and guiding the targeted therapy for patients with kidney renal clear cell carcinoma (KIRC), we analyzed total 526 KIRC samples from The Cancer Genome Atlas (TCGA) database. Our result showed that RIPK2 was upregulated in KIRC tumor samples compared with normal samples. Cox regression was performed to calculate the hazard ratio of RIPK2 expression as an unfavorable prognosis feature for overall survival. Moreover, RIPK2 expression was positively correlated to the high-risk clinical stage, and metastasis features. The upregulation of RIPK2 was strongly correlated with various immune signaling pathway dysregulations as well as immune phenotypes changes in KIRC patient's cohort. In addition, inhibition of RIPK2 activity by either shRNA-mediated knockdown or inhibitor significantly reduced kidney cancer cell viability, trans-migration in vitro, and impaired tumor growth in vivo. In conclusion, elevated RIPK2 expression indicates a worse prognosis for KIRC patients and could serve as a potential prognostic biomarker and therapeutic target in kidney cancer.

MicroRNA-146a limits tumorigenic inflammation in colorectal cancer.

Chronic inflammation can drive tumor development. Here, we have identified microRNA-146a (miR-146a) as a major negative regulator of colonic inflammation and associated tumorigenesis by modulating IL-17 responses. MiR-146a-deficient mice are susceptible to both colitis-associated and sporadic colorectal cancer (CRC), presenting with enhanced tumorigenic IL-17 signaling. Within myeloid cells, miR-146a targets RIPK2, a NOD2 signaling intermediate, to limit myeloid cell-derived IL-17-inducing cytokines and restrict colonic IL-17. Accordingly, myeloid-specific miR-146a deletion promotes CRC. Moreover, within intestinal epithelial cells (IECs), miR-146a targets TRAF6, an IL-17R signaling intermediate, to restrict IEC responsiveness to IL-17. MiR-146a within IECs further suppresses CRC by targeting PTGES2, a PGE2 synthesis enzyme. IEC-specific miR-146a deletion therefore promotes CRC. Importantly, preclinical administration of miR-146a mimic, or small molecule inhibition of the miR-146a targets, TRAF6 and RIPK2, ameliorates colonic inflammation and CRC. MiR-146a overexpression or miR-146a target inhibition represent therapeutic approaches that limit pathways converging on tumorigenic IL-17 signaling in CRC.

Design of pyrido[2,3-d]pyrimidin-7-one inhibitors of receptor interacting protein kinase-2 (RIPK2) and nucleotide-binding oligomerization domain (NOD) cell signaling.

Receptor interacting protein kinase-2 (RIPK2) is an enzyme involved in the transduction of pro-inflammatory nucleotide-binding oligomerization domain (NOD) cell signaling, a pathway implicated in numerous chronic inflammatory conditions. Herein, a pyrido[2,3-d]pyrimidin-7-one based class of RIPK2 kinase and NOD2 cell signaling inhibitors is described. For example, 33 (e.g. UH15-15) inhibited RIPK2 kinase (IC50 = 8 ± 4 nM) and displayed > 300-fold selectivity versus structurally related activin receptor-like kinase 2 (ALK2). This molecule blocked NOD2-dependent HEKBlue NF-κB activation (IC50 = 20 ± 5 nM) and CXCL8 production (at concentrations > 10 nM). Molecular docking suggests that engagement of Ser25 in the glycine-rich loop may provide increased selectivity versus ALK2 and optimal occupancy of the region between the gatekeeper and the αC-helix may contribute to potent NOD2 cell signaling inhibition. Finally, this compound also demonstrated favorable in vitro ADME and pharmacokinetic properties (e.g. Cmax = 5.7 µM, Tmax = 15 min, t1/2 = 3.4 h and Cl = 45 mL/min/kg following single 10 mg/kg intraperitoneal administration) further supporting the use of pyrido[2,3-d]pyrimidin-7-ones as a new structure class of RIPK2 kinase and NOD cell signaling inhibitors.

Receptor-interacting protein kinase 2 contributes to host innate immune responses against Fusobacterium nucleatum in macrophages and decidual stromal cells.

PROBLEM: Chorioamnionitis is caused by a bacterial infection that ascends from the vagina and can cause adverse pregnancy outcomes (APOs). Fusobacterium nucleatum (F. nucleatum) is a periodontal pathogen associated with the occurrence of APOs. In this study, we evaluated whether receptor-interacting protein kinase 2 (Ripk2), an adaptor protein of the cytosolic receptors nucleotide-binding oligomerization domain (NOD)1 and NOD2, in macrophages and human decidual stromal cells (hDSCs) contributes to immune responses against F. nucleatum. METHOD OF STUDY: Bone marrow-derived macrophages (BMDMs) isolated from wild-type (WT) and Ripk2-deficient mice and hDSCs were cultured with F. nucleatum (MOI 1, 10, 100). BMDMs and hDSCs were assessed using enzyme-linked immunosorbent assay, Western blot analysis, real-time PCR, and nitrite assay. RESULTS: Fusobacterium nucleatum-induced production of IL-6, but not of TNF-α and IL-10, was lower in Ripk2-deficient BMDMs than in WT cells. Western blotting revealed a decrease in F. nucleatum-induced p65 phosphorylation in Ripk2-deficient macrophages, whereas mitogen-activated protein kinases activation was comparable between WT and Ripk2-deficient cells. The production of nitric oxide (NO) in response to F. nucleatum and the gene and protein expression of inducible NO synthase was impaired in Ripk2-deficient BMDMs. In hDSCs, F. nucleatum upregulated the gene and protein expression of NOD1, NOD2, and Ripk2 in a time-dependent manner. F. nucleatum also increased the production of IL-6, CXCL8, and CCL2, whereas this production was decreased by the Ripk2 inhibitors SB203580 and PP2. CONCLUSIONS: In conclusion, Ripk2 signaling appears to contribute to the F. nucleatum-induced immune response and can be a preventive and therapeutic target against APOs.

Hepatic NOD2 promotes hepatocarcinogenesis via a RIP2-mediated proinflammatory response and a novel nuclear autophagy-mediated DNA damage mechanism.

BACKGROUND: Key hepatic molecules linking gut dysbiosis and hepatocarcinogenesis remain largely unknown. Gut-derived gut microbiota contains pathogen-associated molecular patterns (PAMPs) that may circulate into the liver and, consequently, be recognized by hepatic pattern recognition receptors (PRRs). NOD2, a general intracellular PRR, recognizes muramyl dipeptide (MDP), present in both gram (+) and gram (-) bacteria. Here, we investigated the role of NOD2 as a molecular sensor translating gut dysbiosis signaling into hepatocarcinogenesis. METHODS: NOD2 expression was measured in clinical hepatocellular carcinoma (HCC) samples using qPCR (80 pairs), western blotting (30 pairs) and immunostaining (141 pairs). The role of NOD2 in hepatocarcinogenesis was examined in the hepatocyte-specific Nod2-knockout (Nod2△hep), Rip2-knockout (Rip2△hep), Lamin A/C-knockout (Lamn△hep) and Rip2/Lamin A/C double-knockout (Rip2/Lamn△hep) mice models of diethylnitrosamine (DEN)/CCl4-induced HCC. RESULTS: NOD2 was upregulated and activated in HCC samples, and high NOD2 expression correlated with poor prognosis in HCC patients. Hepatic NOD2 deletion in vivo decreased DEN/CCl4-induced HCC by reducing the inflammatory response, DNA damage and genomic instability. NOD2 activation increased liver inflammation via RIP2-dependent activation of the MAPK, NF-κB and STAT3 pathways. Notably, a novel RIP2-independent mechanism was discovered, whereby NOD2 activation induces the nuclear autophagy pathway. We showed that NOD2 undergoes nuclear transport and directly binds to a component of nuclear laminae, lamin A/C, to promote its protein degradation, leading to impaired DNA damage repair and increased genomic instability. CONCLUSIONS: We reveal a novel bridge, bacterial sensor NOD2, linking gut-derived microbial metabolites to hepatocarcinogenesis via induction of the inflammatory response and nuclear autophagy. Thus, we propose hepatic NOD2 as a promising therapeutic target against HCC.